1. Field of the Invention
This invention relates to a novel test method for IgA nephropathy. More particularly, the present invention pertains to a rapid and simple test method for IgA nephropathy and a determination method of antibody, which has low risks in emotional distress, peripheral hemorrhage in the kidney and financial burden to the patient, by determining antibody recognizing IgA1 hinge region core peptide in the specimen.
2. Description of Related Art
IgA (immunoglobulin A) nephropathy is a disease concept provided by Berger et al. (J. Urol., 74, pp. 694-695, 1968 ), and is a primary glomerular nephritis having features with clinically poor symptoms except for continuous proteinuria and hematuria, and histological features with precipitants consisting of mainly IgA in the mesangium. The incidence of IgA nephropathy in Japan is high and accounts for 30% of the chronic nephritis. The long term prognosis is not so favorable, and 10-15% of patients with 10 years progress and about 30% of patients with 20 years progress suffer from terminal renal failure. Consequently, IgA nephropathy is especially noticed as a causal disease for terminal renal failure.
At present, the only known test method for IgA nephropathy is the renal biopsy. This test method, however, causes emotional distress for the patients, and further may cause peripheral hemorrhage in the kidney after the biopsy. In addition, the patients must have absolute rest for more than 24 hours after the renal biopsy, and this requires the patients to stay in hospital for several days, and this causes a heavy financial burden for them. Furthermore, the test method has disadvantages including requiring many kinds of test facilities together with long term testing time.
Consequently, a test method for IgA nephropathy is desired, which is simple and operable within a short time as well as giving less emotional distress, without risk of peripheral hemorrhage of kidney and with less financial burden.
An object of the present invention is to provide a rapid and simple test method for IgA nephropathy. Such a test method requires no renal biopsy, which has been essential in the conventional test method for IgA nephropathy. The test method is comprised of detecting antibody, which recognizes the core peptide of the hinge region in IgA1, in specimens. As a result, the patients"" emotional distress, risk of peripheral hemorrhage of the kidney and the financial burden can be reduced.
We have made various trials for solving the above problems and have found a rapid and simple test method for IgA nephropathy and a determination method for antibody, which recognizes the core peptide of hinge region in IgA1, and completed the present invention. Such a test method is comprised of detecting antibody, which recognizes the core peptide of the hinge region in IgA1, in specimens. As a result, the patients"" emotional distress, risk of peripheral hemorrhage of the kidney and the financial burden can be reduced.
The present invention relates to a test method for IgA nephropathy by detecting antibody recognizing the core peptide of the hinge region in IgA1 in specimens. According to preferred embodiments of the present invention, a test method comprising detecting antibody recognizing the core peptide of the hinge acid sequence consisting of region having at least a part of amino acid sequence consisting of
or at least three amino acids thereof. According to a further preferred embodiment of the present invention, the test method comprises using an immobilized solid phase core peptide of the hinge region, said immobilized solid phase of which is microtiter plate or latex particles.
Examples of detection methods for the antibody of the present invention are test method for IgA nephropathy which includes enzyme immunoassay, latex agglutination photometric immunoassay, nephelometric immunoassay, latex slide agglutination test, turbidimetric immunoassay, luminescent immunoassay, fluorescence polarization immunoassay, fluorescence immunoassay and radioimmunoassay.
The present invention is explained in detail with reference to the drawings as follows.
FIG. 1 illustrates a schematic drawing showing the outline of a molecular structure of IgA1.
IgA, which may be involved in a cause for IgA nephropathy, is a type of immunoglobulin. The IgA molecule consists of two heavy chains and two light chains, and is classified into subtypes of IgA1 and IgA2 depending on the difference in heavy chain structures.
In IgA nephropathy, since the deposited IgA in the glomeruli is mainly IgA1 subtype (Conley et al., J. Clin. Invest. 66, pp. 1432-1436, 1980), the properties of IgA1 between those of patients with IgA nephropathy and those of healthy subjects or patients with non-IgA nephropathy may differ from one another.
The largest structural difference between IgA1 and IgA2 exists in the hinge region of the heavy chain. The hinge region of IgA2 is deficient in 13 amino acids (Frangione et al., Proc. Natl. Acad. Sci. USA, 69, pp. 3673-3676, 1972) as compared with the hinge region of IgA1 (SEQ ID NO: 13) [Basic immunology, Upper Vol., pp. 158-162, (1986), The University Tokyo Press].
Baenziger et al. reported that an O-linked sugar chain is bonded to five serine residues of the hinge region in IgA1 molecule (J. Biol. Chem. 249, pp. 7270-7281, 1974).
We have demonstrated that the binding ability between the antibody obtained from rabbit immunized with synthetic peptide of the hinge region in IgA1 and the serum IgA1 of patients with IgA nephropathy was higher than the binding ability between the above antibody and the serum IgA1 of healthy subjects or patients with non-IgA nephropathy. As a result, we have reported that IgA1 of the patients with IgA nephropathy was abnormal in the O-linked sugar chain bound to the hinge region, and in conclusion, the core peptide of the hinge region (core peptide of hinge region means the peptide constructing the hinge region) was in an exposed state (J. Am. Renal. Soc. 8, pp. 538 A, 1997).
The core peptide of the hinge region has structural similarity with mucin insofar as containing large amounts of proline, serine and threonine.
The immunologically activating ability of human mucin core peptide has been known. For example, Kotera et al. reported that the mucin which is frequently expressed in some types of cancer cells has less sugar chain, and, as a result, immune reaction against the exposed core peptide occurs in the host (Cancer Res., 54, pp. 2856-2860, 1994).
We have made extensive studies based on the above findings. As a result, we have found that, in patients with IgA nephropathy, O-linked sugar chain bonded with the hinge region of IgA1 is decreased, and this results in exposing the core peptide of the hinge region, such that an immune reaction against the core peptide of the hinge region in IgA1 is generated and the antibody titer recognizing the core peptide of the hinge region in IgA1 is increased; and that these findings can be applied for testing IgA nephropathy, and completed the present invention.
A rapid test for IgA nephropathy can be performed by determining antibody, which recognizes the core peptide of the hinge region of IgA1, in specimens such as body fluid of the patients. Namely, the present invention relates to a test method for IgA nephropathy based on determining antibody, which recognizes the core peptide of the hinge region in IgA1, in specimens.
In the present invention, the hinge region of IgA1 indicates the region between CH1 domain and CH2 domain in the heavy chains, which construct IgA1 molecule, and may optionally include a structure having added thereto a sequence of Pro-Valxe2x80x94in the N-terminal region.
This region contains disulfide bond between heavy chains, and is rich in proline, serine and threonine.
In the present invention, the core peptide of the hinge region in IgA1 as an antigen can include the peptide constructing the hinge region in IgA1 or a partial structure thereof.
Amino acid sequences of the peptide of the formula (SEQ ID NO: 1),
or peptides having partial structure thereof consisting of at least three amino acids thereof are preferably used. Examples of peptides are peptides comprising amino acid sequences of the formulas:
More preferably, peptides consisting of at least five amino acids, for example, formulas,
ProValProSerThr (SEQ ID NO: 2),
ProSerThrProPro (SEQ ID NO: 3),
ThrProProThrPro (SEQ ID NO: 4),
ProThrProSerPro (SEQ ID NO: 5),
ProSerProSerThr (SEQ ID NO: 6), or
ProSerProSerCys (SEQ ID NO: 7)
can be mentioned.
In the present invention, so far as enabling, all peptides or proteins including peptide constructing the hinge region of IgA1 or a part thereof can be used.
Further, the core peptide of the hinge region in IgA1 modified by sugars, lipids, functional groups, protective groups or cross linking agents, and bound to carrier such as proteins, plastics or latex particles can be used.
In the present invention, any core peptide of the hinge region in IgA1, i.e. antigen, produced by any method, for example, a product by synthesis, a product derived from IgA1 originated from patients or subjects with non-IgA nephropathy, or a product by gene recombinant technology, can be used.
Examples of specimens in the present invention are biological fluid such as blood, plasma, serum, saliva or urine, biological tissue extracts and supernatant solutions of the tissue culture.
Specimens can be used without dilution or with aliquot dilution of buffer solution depending on the principle of detection method, detection sensitivity, detection range and other factors in the present invention.
Specimens can contain anticoagulants such as sodium EDTA, sodium citrate or heparin sodium, materials for stabilizing the specimen, or other substances.
With respect to buffer solutions used in the present invention, buffers which are customarily used in immunological determination methods and preferably have a pH value in the range of from 5 to 9 can be employed.
For example, phosphate buffer, borate buffer, carbonate buffer, Tris-HCl buffer, veronal buffer and Good""s buffer such as HEPES buffer, PIPES buffer and MES buffer, can be used.
For the purpose of preventing non-specific adsorption of proteins to the carrier used in the determination method of the present invention such as microtiter plate and latex, any type of additives, for example, bovine serum albumin (BSA), protein such as gelatin or skimmed milk, surface active agents, sugars, chelating agents, reducing agents or salt can be added in the buffer solution.
The concentration of the additives can be selected from the concentration used in the immunological determination method of antibody, which is applied in known antigen-antibody reactions.
Examples of antibody to be determined in the present invention are all antibodies that react with the core peptide of the hinge region in IgA1 in specimens, and are preferably IgG class, IgM class or IgA class.
In the present invention, a method for immunologically determining the antibody, which recognizes the core peptide of the hinge region in IgA1 as the antigen, in specimens, is characterized by reacting the above described core peptide of the hinge region in IgA1 with the specimen under the condition to form antigen-antibody complex and qualitating or quantitating the antibody, which recognizes the core peptide of the hinge region in IgA1, in the specimen based on assaying the thus generated product of the antigen-antibody reaction.
For example, enzyme immunoassay (EIA) (Proteins, Nucleic Acids and Enzymes, Supplement No. 31, Enzyme immunoassay, pp. 13-26, 1987) can be used.
In one embodiment, a carrier having immobilized thereon core peptide of the hinge region in IgA1 and a specimen are reacted, and a labeled antibody (a complex of anti-human IgG antibody, anti-human IgM antibody, anti-human IgA antibody or fragments thereof having binding activity to antigen and labeled enzyme) is reacted therewith, then the coloring agent (substrate) for developing color by applying the reaction with labeled enzyme bound with the carrier through the antigen-antibody complex, is added and subjected to reaction.
The reaction is terminated, if necessary, by adding enzyme reaction terminator, and optical absorption of the thus generated coloration is measured at a suitable wavelength to quantitatively or qualitatively assay the antibody, which recognizes the core peptide of the hinge region in IgA1, in the specimen.
In a further detailed embodiment, in case that a complex bound with the synthetic core peptide of the hinge region in IgA1 consisting of peptide of at least three amino acids, preferably at least five amino acids, selected from the amino acid sequence of the core peptide of the hinge region in IgA1 such as amino acid sequence of the formula,
and the bovine serum albumin (BSA) at a binding ratio of 1:1-15:1 (peptide:BSA), preferably, 1:1-10:1 (peptide:BSA), is used as an antigen, the antigen adjusted within a concentration range generally from 0.01 xcexcg/ml-100 xcexcg/ml is added to the plastic microtiter plate, generally at 25 xcexcl-200 xcexcl/well, allowed to stand at 4xc2x0 C.-40xc2x0 C., for 10 min.-48 hours washed with suitable buffer to immobilize the antigen. Further, the specimens (such as serum of the patients) diluted with suitable buffer, generally 25 xcexcl-200 xcexcl, are added to the microtiter plate with the immobilized antigen, and allowed to stand generally at 4xc2x0 C.-40xc2x0 C., for 5 min.-48 hours then are washed with suitable buffer.
Next, the self-known labeled antibody (a complex of anti-human IgG antibody, anti-human IgM antibody, anti-human IgA antibody or fragments thereof having binding activity to antigen, and the labeling enzyme, for example generally used in the enzyme immunoassay, such as peroxidase, alkaline phosphatase, xcex2-galactosidase or glucose oxidase) diluted with suitable buffer, generally 25 xcexcl-200 xcexcl, is added and allowed to react generally at 4xc2x0 C.-40xc2x0 C., 10 min.-48 hours then are washed with suitable buffer.
Further, coloring agent (substrate) for developing color with a reaction of the labeled enzyme is added generally 25 xcexcl-200 xcexcl, and is reacted generally within a range at 4xc2x0 C.-40xc2x0 C., for 1 min.-48 hours.
The reaction is terminated, if necessary, by adding enzyme reaction terminator. Optical absorbancy of the supernatant of the enzymatic reaction mixture is colorimetrically measured and the antibody recognizing the core peptide of the hinge region in IgA1 in the specimen is qualitatively or quantitatively measured.
In the above, in case that peroxidase is selected as the labeled enzyme, orthophenylenediamine and the like can be used as a substrate, and the resultant coloring of the enzyme reaction is measured by absorbancy at 490 nm.
Further, in case that alkaline phosphatase is selected as the labeled enzyme, paranitrophenyl phosphate can be used as a substrate, and the resultant color development of the enzyme reaction is measured by absorbancy at 405 nm.
If the antibody titer, which recognizes the core peptide of the hinge region in IgA1 (IgG class antibody titer, IgM class antibody titer or IgA class antibody titer), is high, the above absorbancy values are also high.
For example, the examination for IgA nephropathy can be performed as follows: the absorbancy of the specimens obtained from the healthy subjects or patients with non-IgA nephropathy, or the concentration values in these specimens, which are quantitatively determined by comparing the obtained absorbancy with the calibration curve calculated from the standard substance, are obtained. Measurement above the average of these values+2xc3x97standard deviation are deemed to be positive, and those below or not above the value thereof are considered to be negative. Using these criteria, the examination of subjects, whether or not the patient is suffering from IgA nephropathy, can be determined by detecting the absorbancy of the patients or the quantitative value of concentration obtained from the same manner as above.
The determination method is not limited within the enzyme immunoassay, and all methods, which can determine antibody recognizing the core peptide of the hinge region in IgA1, can be used in the present test method. Examples thereof are as follows:
(a) Conventionally used latex agglutination photometric immunoassay; LAPIA (Sakurabayashi, 1. et al., Nihon Rinsho, Vol. 48, supplement, pp. 13 56-1361, 1990). Latex particles immobilized with the core peptide of the hinge region in IgA1 and a specimen are mixed. Turbidity generated from the result of antigen-antibody reaction is measured as absorbancy. The antibody recognizing the core peptide of the hinge region in IgA1 in the specimen is determined quantitatively or qualitatively based on measurement of changes of the absorbancies.
(b) Nephelometric immunoassay; NIA (Yamagishi, Y. et al., Rinsho-Kensa (Clinical Examinations), Vol. 23, Supplement, pp.1286-1289, 1979). Latex particles immobilized with the core peptide of the hinge region in IgA1 are mixed with a specimen. The thus produced antigen-antibody complex is irradiated with light (laser) and changes of the scattering strength are measured, and the antibody recognizing the core peptide of the hinge region in IgA1 in the specimen is quantitatively or qualitatively measured.
(c) Latex slide agglutination (Shibata, S. et al., Equipment and Reagents, Vol. 11, pp. 338-342, 1988). Latex particles immobilized with the core peptide of the hinge region in IgA1 and a specimen are mixed on the plate, and agglutination of latex particles generated as a result of antigen-antibody reaction is confirmed macroscopically to qualitate the antibody, which recognizes the core peptide of the hinge region in IgA1, in the specimen.
(d) Counting immunoassay; CIA(Hashimoto, K. et al., Test and Technology, Vol. 22, No. 5, pp. 67-68, 1994). Latex particles immobilized with the core peptide of the hinge region in IgA1 and a specimen are mixed. Sizes and numbers of aggregates generated as a result of antigen-antibody reaction are measured. The antibody recognizing the core peptide of the hinge region in IgA1 in the specimen can be qualitatively or quantitatively measured.
(e) Turbidimetric immunoassay; TIA (Toyama, T., Clinical pathology, Vol. 35, pp. 868-873, 1987). The core peptide of the hinge region in IgA1 and a specimen are mixed. Turbidity generated as a result of antigen-antibody reaction is measured and changes of absorbancy are measured to qualitate or quantitate the antibody recognizing the core peptide of the hinge region in IgA1 in the specimen.
(f) Luminescent immunoassay; LIA (xe2x80x9cProteins, Nucleic Acids and Enzymesxe2x80x9d, Suppl. 31, xe2x80x9cEnzyme Immunoassayxe2x80x9d, pp. 252-263, 1987).
(g) Fluorescence polarization immunoassay; FPIA (Kurata, K., xe2x80x9cNew case studies on immunoassay and application for development of diagnostic reagents and therapeutic agentsxe2x80x9d, Keiei-Kyoiku Publ., Jun. 20, 1985, pp. 91-102).
(h) Immunoelectrophoresis (Urushizaki, I. et al., xe2x80x9cNew case studies on immunoassay and application for development of diagnostic reagents and therapeutic agentsxe2x80x9d, Keiei-Kyoiku Publ., Jun. 20, 1985, pp. 63-72).
(i) Spin immunoassay; SIA (Sago, H., xe2x80x9cNew case studies on immunoassay and application for development of diagnostic reagents and therapeutic agentsxe2x80x9d, Keiei-Kyoiku Publ., Jun. 20, 1985, pp. 52-62).
(j) Fluorescence immunoassay; FIA (Hashimoto, T. et al., Test and Technology, Vol. 22, No. 5, pp. 61-66, 1994), and
(k) Radioimmunoassay; RIA (Kurata,K., xe2x80x9cNew case studies on immunoassay and application for development of diagnostic reagents and therapeutic agentsxe2x80x9d, Keiei-Kyoiku Publ., Jun. 20, 1985, pp. 33-41).
Qualitative and quantitative assay methods for a product of antigen-antibody reaction in the present invention are not limited to those performed by manual operation but can be performed by applying automatic analysis.
In the enzyme immunoassay,in case of determining antibody recognizing the core peptide of the hinge region in IgA1 in specimens using microtiter plate immobilized with the core peptide of the hinge region in IgA1, for example, BECKMAN Biomek 1000 Automated Laboratory Workstation (Beckman Inc.) or CELL ASSAY-2000 ROBOTIC ASSAY SYSTEM (Moritex Inc.), both of which are automated system equipment with supplying specimens, supplying reagents, washing operation, measurement of absorbancy and data processing, can be used.
In detail, a specimen, enzyme labeled antibody and substrate solution are added, in this order, together with washing operation among each addition, in the microtiter plate immobilized with the core peptide of the hinge region in IgA1, and the reaction is terminated, if necessary, by adding a terminator for enzyme reaction, then absorbancy of the color development as a result of the enzyme reaction between the labeled enzyme and substrate used is measured at appropriate wave length selected from the customary measurement conditions to determine the antibody recognizing the core peptide of the hinge region in IgA1 in the specimen.
In the latex agglutination photometric immunoassay, in order to determine antibody, which recognizes the core peptide of the hinge region in IgA1 in specimen by using latex particles having immobilized thereon the core peptide of the hinge region in IgA1, for example, a Hitachi 705, 7050, 7150, 736 or 7070 automatic analyzer, which are automatic equipment systems for supplying specimens, supplying reagents, measurement of absorbance and data processing, can be used.
In detail, latex particles immobilized with the core peptide of the hinge region in IgA1 and a specimen are mixed in a reaction cell, and the blank is corrected by measuring with suitable wave length selected from the known measurement conditions, preferably at 400 nm-950 nm, then changes of absorbancy after a constant time are measured to determine antibody recognizing the core peptide of the hinge region in IgA1 in the specimen.
In the method of the present invention, measurement operations may be facilitated if the necessary reagents for practicing the present invention are prepared in a kit. Further, it is convenient to practice the present invention using automated analytical equipment.
Example of reagent kits used in the present invention is a kit comprising a combination of a carrier (such as microtiter plate or latex particles) immobilized with the core peptide of the hinge region in IgA1 and reagents necessary for practice in the present invention such as buffer, standard substance, labeled antibody, substrate, solubilizing agent for substrate or reaction terminator.